Chaperone Recycling in Late-Stage Flagellar Assembly

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.     

鞭毛细菌游动机理研究进展

鞭毛细菌是在低雷诺数条件下运动的生物体,通过旋转鞭毛而向前游动。通过概述细菌鞭毛的游动机理、在不同游动方式下鞭毛丝几何形态之间转换的物理现象、菌体反转对成束的影响以及在靠近壁时运动的墙效应机理的最新研究,提出一种与鞭毛细菌运动动力相似的宏观等效模型,该模型可以用于进一步研究鞭毛细菌的游动机理,并为仿生微型游动机器人的构造和应用提供理论研究的基础。     

CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。     

CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

Isolation of the Flagellar Swelling and Identification of Retinal in the Phototactic Flagellate, Ochromonas danica (Chrysophyceae)

Ochromonas danica, a freshwater, planktonic chrysophyte, is capable of sensing the light conditions of its environment. This biflagellate alga has a swelling near the base of the short flagellum and a chloroplastidic stigma in close association with it. A procedure is described for the isolation of this three dimensional flagellar swelling, the presumed photoreceptor. In contrast to an earlier method developed for the isolation of the paraflagellar swelling from Euglena gracilis, the protocol reported here for Ochromonas results in higher yields that should facilitate future biochemical investigations and could open avenues of investigation for the isolation and purification of the presumptive receptor protein. To verify the hypothesis that a rhodopsin-like protein might be present in this alga, we applied a standard extraction procedure successfully used in the identification of retinal. We here report the purification and identification of all-trans retinal in Ochromonas cells by column chromatography, HPLC and GC-MS. Since retinal is the chromophore of rhodopsin-like proteins, this finding may suggest that in these unicellular algae, too, a rhodopsin-like protein could be the photoreceptor pigment.     

Green flagellar autofluorescence in brown algal swarmers and their phototactic responses

A flavin-like green autofluorescent substance is noticed to occur in one of the flagella of flagellated cells in the Phaeophyceae, Chrysophyceae, Synurophyceae, Xanthophyceae and Prymnesiophyceae. In the phaeophycean swarmers the autofluorescence occurs in the posterior flagellum throughout its length. It is considered to be involved in the photoreception of phototaxis, since it almost always occurs in the swarmers which have a flagellar swelling and stigma and show phototaxis. In the phaeophycean swarmers, the stigma is shown to act as a concave reflector mirror focusing the reflection light onto the flagellar swelling. In the action spectrum studies, phaeophycean swarmers showed phototaxis between 370 and 520 nm, having two major peaks at 420 or 430 nm and 450 or 460 nm. Their responses were true phototactic and not photophobic. Rotation of the swarmer was shown to be essential in the photoreception ofEctocarpus gametes. Recipient of the Botanical Society Award for Young Scientists, 1991.     

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca²⁺ concentration ([Ca²⁺]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A ⁴⁵Ca²⁺-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH₄Cl treatment. NH₄Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.     

Flagellar movement of human spermatozoa

Flagellar movement of human spermatozoa held by their heads with a micropipette was recorded by means of a video-strobe system. Spermatozoa were studied in normal Hanks' solution, Hanks' solution with increased viscosity, cervical mucus, and hyaluronic acid. When flagellar movement in normal Hanks' solution was observed from the direction parallel to the beating plane, segments of the flagellum in focus did not lie on a straight line but on two diverging dashed lines. The distance between the two dashed lines was about 20% of the bend amplitude in the major beating plane. These observations indicate that flagellar beating of human spermatozoa in normal Hanks' solution is not planar. In contrast, segments of the flagellum in focus lay on a straight line when the spermatozoa were observed in Hanks' solution with increased viscosity, cervical mucus, or hyaluronic acid. In normal Hanks' solution, free swimming spermatozoa rotated constantly around their longitudinal axes with a frequency similar to the beat frequency, whereas little or no rotation of spermatozoa occurred in Hanks' solution with increased viscosity, in cervical mucus, or in hyaluronic acid. We conclude that human spermatozoa in normal Hanks' solution beat with a conical helical waveform having an elliptical cross section, the semiaxes of which have a ratio of 0.2. The three-dimensional geometry of the flagellar movement is responsible for the rotation of the sperm around their longitudinal axes.     

Flagellar apparatus ultrastructure inMesostigma viride (Prasinophyceae)

The ultrastructure of the flagellar apparatus ofMesostigma viride Lauterborn (Prasinophyceae) has been studied in detail with particular reference to absolute configurations, numbering of basal bodies, basal body triplets and flagellar roots. The two basal bodies are interconnected by three connecting fibers (one distal fiber = synistosome, and two proximal fibers). The flagellar apparatus shows 180° rotational symmetry; four microtubular flagellar roots and two system II fibers are present. The microtubular roots represent a 4-6-4-6-system. The left roots (1s, 2s) consist of 4 microtubules, each with the usual 3 over 1 root tubule pattern. Each right root (1d, 2d) is proximally associated with a small, but typical multi-layered structure (MLS). The latter displays several layers corresponding to the S₁ (the spline microtubules: 5–7), and presumably the S₂—S₄ (the lamellate layers) of the MLS of theCharophyceae. At its proximal origin (near the basal bodies) each right root originates with only two microtubules, the other spline microtubules being added more distally. The structural and positional information obtained in this study strongly suggest that one of the right roots (1d) ofMesostigma is homologous to the MLS-root of theCharophyceae and sperm cells of archegoniate land plants. Thus the typical cruciate flagellar root system of the green algae and the unilateral flagellar root system of theCharophyceae and archegoniates share a common ancestry. Some functional and phylogenetic aspects of MLS-roots are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

喜树幼苗中喜树碱和10-羟基喜树碱对热激的响应特点和意义

植物在长期的生态环境适应过程中,产生了包括生物碱在内的大量次生代谢物。本文以我国特有树种——喜树(Camptotheca acuminata Decaisne)为材料,研究其不同器官中喜树碱(camptothecin,CPT)和10-羟基喜树碱(10-hydroxycamptothecin,HCPT)在不同热激温度和时间情况下的含量变化。CPT和HCPT变化呈现出较好的相互消长关系,并且分别在38℃和40℃达到各自的峰值,比以丙二醛和叶绿素为指标的致死温度低了2-4℃：HCPT在热激过程中的变化较CPT活跃;极易受到攻击和伤害的嫩叶中的生物碱含量变化最大。由此推断,CPT和HCPT遵循“幼嫩和生殖器官优先保护”的原则,从而有效地缓解了高温胁迫,并且HCPT和CPT代表了不同的防御策略。     

Flagellar regeneration and associated scale deposition inPyramimonas gelidicola (Prasinophyceae,Chlorophyta)

Summary During regeneration of mechanically amputated flagella, flagellar scales and the subtending membrane accumulate in a villiform scale reservoir in which the scales interact to form patterns on the villi reminiscent of the arrangement they later assume on the flagellum. The reservoir membrane is continuous with the plasmalemma, and the scales, attached directly and indirectly to the membrane, leave the reservoir and migrate toward the developing flagella where they assemble into highly ordered layers. It is proposed that scale-scale interactions induce a process of auto-assembly initiating the complex arrangement of scale tiers on the flagellum and cell body.     

喜树幼苗中喜树碱和10-羟基喜树碱对热激的响应特点和意义

植物在长期的生态环境适应过程中,产生了包括生物碱在内的大量次生代谢物.本文以我国特有树种--喜树(Camptotheca acuminata Decaisne)为材料,研究其不同器官中喜树碱(camptothecin,CPT)和10-羟基喜树碱(10-hydroxycamptothecin,HCPT)在不同热激温度和时间情况下的含量变化.CPT和HCPT变化呈现出较好的相互消长关系,并且分别在38℃和40℃达到各自的峰值,比以丙二醛和叶绿素为指标的致死温度低了2~4℃;HCPT在热激过程中的变化较CPT活跃;极易受到攻击和伤害的嫩叶中的生物碱含量变化最大.由此推断,CPT和HCPT遵循"幼嫩和生殖器官优先保护"的原则,从而有效地缓解了高温胁迫,并且HCPT和CPT代表了不同的防御策略.     

Light-Induced Body Movement of Euglena gracilis Coupled to Flagellar Photophobic Responses by Mechanical Stimulation*†

SYNOPSIS. In low viscosity media, Euglena gracilis strain Z responds to a sudden change in light intensity by a cessation of forward movement, followed by a reorientation of the locomotor flagellum which results in turning of the cell around the lateral axis (photophobic response). At a viscosity interface between low [～ 1 cP (centipoise)] and high (4000 cP) media, the cells exhibit avoidance responses or become immobilized in the higher viscosity medium. Upon changing the light intensity, free swimming cells have photophobic responses, while immobilized ones undergo body contractions. For cells immersed in media of varying viscosity, the delay between light stimulation and body contraction (transduction time) is shortest at high viscosities. From 500 to 2000 cP, where the cells are capable of both movement and light-induced body contractions, there is a logarithmic dependence of the transduction time on the viscosity. The transduction time does not vary appreciably with the intensity of the primary light stimulus within a range of 0.14-1.13 kW/m².     

Rhodopsin Receptors of Phototaxis in Green Flagellate Algae

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.     

Flagellar transformation in the heterokontEpipyxis pulchra (Chrysophyceae): Direct observations using image enhanced light microscopy

Summary Cells ofEpipyxis pulchra possess two heteromorphic flagella that differ markedly in function, particularly during motility and prey capture. Flagellar heterogeneity is achieved during the course of at least three cell cycles. Prior to cell division, cells produce two new long, hairy flagella while the parental long flagellum is transformed into a new short, smooth flagellum. The parental short flagellum remains a short flagellum for this and subsequent cell division cycles. Although flagellar transformation requires only two cell cycles, developmental differences exist between daughter cells and the maturation of a flagellum/basal body requires at least three cycles.     

Chlororespiration in Green Algae Isolated From Desert Crusts

Photosynthetic organisms enduring extreme temperatures, low water availability, or high light require photoprotective mechanisms to prevent sustained damage to photosynthetic machinery. Green microalgae living in desert crust communities of the south‐western US experience all these environmental stresses, yet photophysiological studies of green algae in the literature have focused on only a handful of common aquatic and marine species. We are examining the variation in green algal photoprotective mechanisms that is the result of natural selection acting independently in multiple lineages of highly diverse desert green algae (Chlorophyta) within the classes Chlorophyceae and Trebouxiophyceae. We have found that unusually extensive dark reduction of the plastoquinone pool is a prominent photophysiological feature among these desert algae; this reduction may be linked with enhanced chlororespiration. Recently, chlororespiration in higher plants has been linked through mutant analysis to control of the carotenoid synthetic pathway, heat stress, and starch metabolism among other pathways, though the function of chlororespiration remains controversial. Given that green algae and higher plants are monophyletic, analysis of potential chlororespiration in desert green algae may help decipher the evolution of the chlororespiratory process as well as its potential role in photoprotection in desert habitats.     

绿藻[FeFe]氢化酶

该文介绍了绿藻[FeFe]氢化酶的研究现状,包括酶的结构、催化中心、金属簇的性质,以及对氧的敏感性和可能的解决办法。并且对已报道的绿藻[FeFe]氢化酶基因及其调控等问题作了介绍。     

Flagellar waveforms of gametes in the brown alga Ectocarpus siliculosus

Brown algae are members of the Stramenopiles and their gametes generally have two heterogeneous flagella: a long anterior flagellum (AF) with mastigonemes and a short posterior flagellum (PF). In this study, swimming paths and flagellar waveforms in free-swimming and thigmotactic-swimming male and female gametes and in male gametes during chemotaxis, were quantitatively analysed in the model brown alga Ectocarpus siliculosus. This analysis was performed using a high-speed video camera. It was revealed that the AF plays a role in changing the locomotion of male and female gametes from free-swimming to thigmotactic-swimming and also in changing the swimming path of male gametes from linear to circular during chemotaxis. In the presence of a sex pheromone, male gametes changed their swimming path from linear (swimming path curvature, 0–0.02 µm^–1) to middle and small circular path (swimming path curvature, 0.04–0.20 µm^–1). The flagellar asymmetry and the deflection angle of the AF became larger, whereas the oscillation pattern of the AF was stable. However, there was no correlation between the flagellar asymmetry and the deflection angle of the AF and the path curvature when the male gametes showed middle to small circular paths. The PF irregularly changed the deflection angle and the oscillation pattern was unstable depending on the gradient of the sex pheromone concentration. AF waveforms were independent of PF locomotion during chemotaxis. This means that the AF has the ability to change the swimming path of male gametes – for example, from a highly linear path to a circular path – while changes in locomotion from a middle circle path to a small circle path is the result of beating of the PF.     

Electrophysiology of Turgor Regulation in Marine Siphonous Green Algae

We review electrophysiological measures of turgor regulation in some siphonous green algae, primarily the giant-celled marine algae, Valonia and Ventricaria, with particular comparison to the well studied charophyte algae Chara and Lamprothamnium. The siphonous green algae have a less negative plasma membrane potential, and are unlikely to have a proton-based chemiosmotic transport system, dominated by active electrogenic K⁺ uptake. We also make note of the unusual cellular structure of the siphonous green algae. Hypertonic stress, due to increased external osmotic pressure, is accompanied by positive-going potential difference (PD), increase in conductance, and slow turgor regulation. The relationship between these is not yet resolved, but may involve changes in K⁺ conductance (G _K) or active K⁺ transport at both membranes. Hypotonic turgor regulation, in response to decreased external osmotic pressure, is ∼3 times faster than hypertonic turgor regulation. It is accompanied by a negative-going PD, although conductance also increases. The conductance increase and the magnitude of the PD change are strongly correlated with the magnitude of hypotonic stress.     

Post-transcriptional Control of Nitrate Reductase Formation in Green Algae

Cycloheximide (2·0 µg ml^–1) inhibits theincorporation of [¹⁴C]phenylalanine and [¹⁴C]adenine into insolublecompounds in Ankistrodesmus braunii. 6-Methylpurine (1·0mM) inhibits only the incorporation of [14C]adenine and it isconcluded that it inhibits RNA synthesis. When ammonium-growncells of Ankistrodesmus or Chlorella are nitrogen-starved orwhen ammonium-grown cells of Dunalitlla are resuspended in nitratemedium, the appearance of nitrate reductase in these organismsis not inhibited by 6-methylpurine. The appearance of nitratereductase activity in Ankistrodesmus or Chlorella is inhibitedby 6-methylpurine when ammonium-grown organisms are preincubatedwith this substance for 1-2 h before nitrogen starvation. Itis concluded that cells growing with ammonium and lacking nitratereductase activity nevertheless contain preformed mRNA for nitratereductase synthesis.